Double-stranded RNA based interference (dsRNAi) has become an important tool for reverse functional genomics (Fire 1998). RNAi is a naturally occurring defense mechanism that is highly conserved among eukaryotes. RNAi protects the genome against invention by mobile genetic elements, such as transposons, viruses, and other highly repetitive genomic sequences, and also to control the function of developmental programs in eukaryotic organisms (Sidahmed 2010).
RNAi involves the cleavage of double-stranded RNA (dsRNA) by an RNaseIII-type enzyme called Dicer into small interfering RNAs (siRNA), which then direct sequence-specific, homology-dependent, post-transcriptional gene silencing by binding to their complementary RNA sequences and triggering their elimination through degradation or by inducing translational inhibition (Fire 1998; Meister 2004).
Multivalent RNA (MV-RNA) represents a junction-class RNA molecule that is not canonical dsRNA, but which has a similar mode of action to dsRNA-based RNAi molecules described above. Uniquely, MV-RNA exhibits the ability to cleave multiple sites on the same or different genes simultaneously as well as utilize different pre-processing pathway than dsRNAi (U.S. Patent Publication No. 2011/0159586 and PCT Publication No. WO2012/014155) (FIG. 15).
RNAi molecules such as siRNA, shRNA, miRNA or MV-RNA interact with Ago, PAZ, and PIWI domains as initial steps in loading into the RNA Induced Silencing Complex (RISC). Thus, controlling the accessibility of the 5′ and 3′ ends of the RNAi molecule by the silencing complex (i.e. RISC) or even Dicer would enhance specificity. Additionally, the production of multiple siRNA molecules from the biogenesis of longer dsRNA by Dicer is a means of producing multiple siRNA molecules from a single transcript. Cleavage of dsRNA RNAi pre-cursors by Dicer or Drosha endonucleases is common in plants, animals, and humans. However, long dsRNA is a poor RNAi trigger in mammals due to the negative immunological response, is rapidly degraded in nearly all uses, and does support the precise production of multiple short RNAi molecules, such as MV-RNA, from a single transcript.
RNA nanotechnology itself has been around since 1998. Many efforts have been made over the years to overcome the susceptibility of RNA to nuclease degradation, structural flexibility, serum instability, and RNase sensitivity and the challenges remain for most commercial uses when building concrete shapes with RNA. Several nucleic acid self-assembly methods, including the use of structural DNA scaffolds, have been employed to generate siRNA-containing nanostructures for in vivo delivery.
Utilizing the intermolecular interactions of RNA, diverse RNA assemblies of nanoparticles have been tried. The pRNA dimer, trimer, and hexamer formations (Guo 1987, 1988; Shu 2004, 2007, 2011, Haque 2012) have also been well studied. The pRNA molecules contain the bacteriophage phi29 at their core, and one to many active modulating molecules at each end of the 3-way junction. In vitro and in vivo results have shown that the pRNA substrate can be directed by RNA, DNA aptamer or Peptide ligand and be gene modulating by appended siRNA, shRNA, ribozyme, peptide, or antibody. RNA nanorings based on RNAI/II inverse kissing complexes (Yingling and Shapiro 2007; Afonin et al. 2011; Grabow et al. 2011); kissing loops of HIV RNA (Chang and Tinoco 1994; Bindewald et al. 2008) and the hand-in-arm interactions of Drosophila bicoid mRNA (Wagner et al. 2004); (2) palindrome sequence-mediated formation of pRNA dimers, tetramers, and arrays (Shu et al. 2004); (3) RNA motifs as LEGO pieces to build quaternary structures via non-templated assemblies including tecto-RNA, two-way junctions (2WJs), 3WJs, and four-way junctions (4WJs), and self-assembly by colE1 kissing loop interactions (Prats et al. 1990; Clever et al. 1996; Mujeeb et al. 1998; Jaeger and Leontis 2000; Lilley 2000; Shu et al. 2011a; Haque et al. 2012); (4) extension of arms of thermodynamically stable core to carry multiple therapeutic small RNAs (Shu et al. 2011a; Haque et al. 2012); (5) use of RNA binding proteins to serve as scaffolds for the formation of nanostructures, such as equilateral triangle constructs, where three proteins are bound to an RNA scaffold containing a kink-turn motif for protein binding (Schroeder et al. 2010; Ohno et al. 2011).
Despite nearly 30 years of study, each RNA nanoparticle is handicapped by features making commercial use difficult. Nanorings are dependent on non-covalent kissing loop interactions that can denature easily in temperature gradients; are not able to be formed efficiently in vivo; and the rational assembly can be variable. The pRNA overcomes the stability issues of Nanorings, but lack the molarity by being limited to three active molecules and also lack a rational control of nuclease degradation. In fact, nearly all nanoparticles above are either limited by non-covalent bonding, molarity limits, or by the lack of nuclease control.
It was previously shown that RNA Microsponge particles could be made by in vitro Rolling Circle Transcription and even used in RNAi with little or no toxicity (Hammond 2012). By utilizing a canonical shRNA structure expressed repetitively as a single stranded concatamer, spherical particles of 2 μM are formed and then later condensed by PEI treatment to ˜200 nanometers. Hammond illustrated that the transcription of hundreds of thousands of shRNA form sheets that eventually collapse into spherical form—referred to as “microsponges.” Such microsponges are also shown to be active RNAi triggers. However in 2014, Hammond proved that such spherical formation was unrelated to the RNA itself and was the result of the RNA binding to nanocrystalline magnesium pyrophosphate during the T7 transcription reaction. While such RNA microsponges can be formed and even used in RNAi, there lacks the ability to produce smaller sizes of a programmed composition as well as the ability to do so in vivo.
Spherical Nucleic Acid (SNA) nanoparticle conjugates have also been published recently (Zheng 2012, 2013; Zhou 2013, Jensen 2013, Ding 2014) showing conjugated siRNA arranged spherically around a gold particle. Gold nanoparticles offer both covalent and non-covalent attachment of the active nucleic acid molecule. The arrangement is stacked around the gold particle center. While the approach has proven to be active due to the spherical arrangement of the nucleic acids and cellular penetration, it remains a synthetic (inorganic) delivery vector.
Viral coat proteins or capsid proteins function in the transportation and protection of nucleic acids. It was shown half a century ago that infective virus particles of helical symmetry self-assemble upon mixing aqueous solutions of the coat protein and RNA (H. Fraenkel-Conrat, 1955). In most cases, this protective layer is due to the presence of multiple copies of a coat protein that self-assemble into what is typically rod or sphere-like shapes surrounding the nucleic acid. While many of the details surrounding the spontaneous self-assembly process remain obscure, recent data (see citations ‘Coat Protein References’) suggests that at least the protein-protein interactions and the nucleic acids characteristics dictate the structural outcome. In the case of Cowpea Chlorotic Mottle Virus (CCMV), evidence suggests that the diameter is controlled by nucleotide length. Researchers determined that a length of less than 3000 nt resulted in a ˜24-26 nm Coat Protein (CP) diameter, and that a length greater than 4,500 nt resulted in a ˜30 nm Coat Protein (CP) diameter when combined with a protein/RNA mass ratio of 6:1. While the use of CP in vitro and in vivo has been demonstrated to encapsulate nucleic acids, this RNA length to CP dependency is inefficient for long dsRNA uses and not possible for short RNAi triggers without pre-packaging (i.e., lipids).
There remains a need for methods and compositions that allow for self-forming polynucleotide nanoparticles for gene modulation with programmable diameters, nuclease stability, molarity, cell-specificity, uptake, and reliable nuclease biogenesis of the active trigger—that is useful for both transgenic and exogenic uses. The present invention addresses this need, and can be applied in humans, animals, plants, insects, and fungi.